Abstract
Although recent studies reveal that activation of the metabolic and Ca2+ sensor AMPK strongly inhibits smooth muscle contraction, there is a paucity of information about the potential linkage between pharmacological AMPK activation and vascular smooth muscle (VSM) contraction regulation. Our aim was to test the general hypothesis that the allosteric AMPK activator A-769662 causes VSM relaxation via inhibition of contractile protein activation, and to specifically determine which activation mechanism(s) is(are) affected. The ability of A-769662 to cause endothelium-independent relaxation of contractions induced by several contractile stimuli was examined in large and small musculocutaneous and visceral rabbit arteries. For comparison, the structurally dissimilar AMPK activators MET, SIM, and BBR were assessed. A-769662 displayed artery- and agonist-dependent differential inhibitory activities that depended on artery size and location. A-769662 did not increase AMPK-pT172 levels, but did increase phosphorylation of the downstream AMPK substrate, acetyl-CoA carboxylase (ACC). A-769662 did not inhibit basal phosphorylation levels of several contractile protein regulatory proteins, and did not alter the activation state of rhoA. A-769662 did not inhibit Ca2+- and GTPγS-induced contractions in β-escin-permeabilized muscle, suggesting that A-769662 must act by inhibiting Ca2+ signaling. In intact artery, A-769662 immediately reduced basal intracellular free calcium ([Ca2+]i), inhibited a stimulus-induced increase in [Ca2+]i, and inhibited a cyclopiazonic acid (CPA)-induced contraction. MET increased AMPK-pT172, and caused neither inhibition of contraction nor inhibition of [Ca2+]i. Together, these data support the hypothesis that the differential inhibition of stimulus-induced arterial contractions by A-769662 was due to selective inhibition of a Ca2+ mobilization pathway, possibly involving CPA-dependent Ca2+ entry via an AMPK-independent pathway. That MET activated AMPK without causing arterial relaxation suggests that AMPK activation does not necessarily cause VSM relaxation.
Highlights
Contraction of vascular smooth muscle (VSM) is highly dependent on the degree of myosin light chain (MLC) phosphorylation which, in turn, is dependent on an elevation in [Ca2+]i leading to increases in MLC kinase (MLCK) activity, and on rhoA kinase (ROCK)- and protein kinase C (PKC)- dependent inhibition of MLC phosphatase activity (Guibert et al, 2008; Puetz et al, 2009)
To determine whether 5 -adenosine monophosphate kinase (AMPK) α2 and especially AMPK β1 subunits are expressed in arteries, the relative expression of these subunits was examined by comparing α1/α2 and β1/β2 expression ratios in muscular arteries (FA, epigastric artery (EA), renal artery (RA), and mesenteric artery (MA)) and the large elastic carotid artery (CA) using Western blot analysis
The low α1/α2 expression of arterial muscle was not due to the inability of α1 antibody to detect α1 because, using the same antibodies and ratiometric analysis, we found that mouse ileum expressed nearly 100% α1, and that mouse extensor digitorum longus (EDL) expressed nearly 100% α2 (Figure 2C)
Summary
Contraction of VSM is highly dependent on the degree of myosin light chain (MLC) phosphorylation which, in turn, is dependent on an elevation in [Ca2+]i leading to increases in MLC kinase (MLCK) activity, and on rhoA kinase (ROCK)- and PKC- dependent inhibition of MLC phosphatase activity (Guibert et al, 2008; Puetz et al, 2009). Small arteries (i.e., mouse saphenous artery) appear to respond less well than large arteries (i.e., mouse aorta) (Davis et al, 2012), suggesting heterogeneity of VSM responsiveness to AMPK within different regions of the vascular tree. Arteries along the vascular tree can be divided into large elastic (e.g., aorta and carotid), large muscular (e.g., femoral and renal), small muscular (e.g., branching arteries such as second to fourth order mesenteric and epigastric) and the still smaller arterioles (Ratz, 2016). No study has yet compared the effects of A-769662 in large and small muscular arteries that display functionally distinct responses to contractile stimuli
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